Proc. Natl. Acad. Sci. USAVol. 85, pp. 8649-8653, November 1988Medical Sciences

Rapid activation of protein kinase C in isolated rat liver nuclei byprolactin, a known hepatic mitogen

(prolactin receptor/dose dependence/anti-prolactin antiserum inhibition/anti-prolactin receptor antibody inhibition)

ARTHUR R. BUCKLEY, PAUL D. CROWE, AND DIANE HADDOCK RUSSELLDepartment of Pharmacology and Therapeutics, University of South Florida College of Medicine, Tampa, FL 33612

Communicated by Robert A. Good, July 22, 1988

ABSTRACT Rat liver nuclei pure by enzymatic and elec-tron microscope criteria contain protein kinase C (PKC) thatcan be activated several hundredfold within 3 min of additionof prolactin or phorbol 12-tetradecanoate 13-acetate. Ratprolactin stimulated PKC maximally at 10-12 M, whereasovine prolactin was maximally stimulatory at 10-1o M. Acti-vation was time and dose dependent, exhibited a biphasicpattern, and was blocked by anti-prolactin antiserum, by PKCinhibitors such as 1-(5-isoquinolinylsulfonyl)-2-methylpipera-zine (H-7) and sphingosine, and by cyclosporine. Moreover, theability of prolactin to activate nuclear PKC was inhibitedtotally by a monoclonal antibody to the rat liver prolactinreceptor, implicating a prolactin receptor-mediated activationprocess. Epidermal growth factor (EGF), a liver mitogen,caused a lesser but significant activation of nuclear PKC.However, EGF and suboptimal prolactin were synergistic.Human growth hormone, which has lactogenic properties,stimulated PKC activity, whereas nonlactogenic substancessuch as ovine growth hormone, insulin, dexamethasone, and8-bromo-cAMP were inactive. That this may be a generalmechanism for prolactin is suggested by the ability of prolactinto stimulate PKC 140-fold in rat splenocyte nuclei. Prolactinhas comitogenic properties in lymphocytes.

Prolactin is an adenohypophyseal polypeptide hormonethought to exert its effects on macromolecular synthesis atthe level of the cell nucleus and Golgi complex after receptor-mediated endocytosis (1, 2). Administration of prolactin torats induces hepatic ornithine decarboxylase and plasmino-gen activator (3-5), specific enzyme markers expressed earlyduring the G1 phase of the cell cycle, and stimulates DNAsynthesis and subsequent hepatomegaly (6, 7). The effects ofprolactin on hepatic cell cycle progression mimic those inresponse to partial hepatectomy (8), a response regulated bycirculating factors, as demonstrated in the classic parabioticexperiments of Moolten and Bucher (9). Tumor promoterssuch as phorbol 12-tetradecanoate 13-acetate (PTA) alsoinduce ornithine decarboxylase and plasminogen activator inthe liver and other organs such as the skin (10-12). Further,prolactin has tumor-promoting properties in carcinogen-exposed rat liver (7). Thus, it seemed likely that prolactinmight exert its cellular action through the activation ofprotein kinase C (PKC).

Prolactin administration to rats results in a rapid activationof hepatic PKC with a characteristic elevation of activity inthe particulate fraction (13). The direct activation ofPKC bythe addition ofPIA (14) elevated particulate PKC activity ina manner similar to prolactin. In addition, partial hepatec-tomy triggered a rapid increase in circulating prolactin (within1 min) and an elevation of PKC activity in the particulatefraction of the remaining liver (13). These data provide

evidence that prolactin-dependent intracellular signaling in-volves the activation of PKC. Other studies have shown thatPTA is capable of stimulating mitogenesis in a prolactin-dependent cell line, the Nb2 node lymphoma cell line (15, 16),and inhibitors of PKC block prolactin-stimulated prolifera-tion in this cell line and affect prolactin-requiring processesin other systems (17, 18).The recent characterization of the rat liver prolactin

receptor by Kelly and coworkers (19) demonstrated a struc-ture similar to that ofreceptors that are primarily transportersof molecules (such as transferrin). Concurrently, other work-ers reported nuclear PKC detected both immunochemicallyand by its calcium and phospholipid activity requirements inliver (20) and in NIH 3T3 cells (21). In lymphocytes, a nuclearlamina protein, lamin B, was rapidly phosphorylated afterPKC activation with PTA (22).The possibility of prolactin receptors at the nucleus (1, 2),

the presence of PKC in rat liver nuclei and subnuclearfractions (20), and the ability ofPKC to influence the nucleartranscription of specific genes (23, 24), including the ornithinedecarboxylase gene(s) (23), led us to explore whether pro-lactin could activate nuclear PKC. We found a rapid several-hundredfold activation ofPKC in rat liver nuclei in responseto prolactin that was dose dependent and could be blocked byrat anti-prolactin receptor antibody and by anti-prolactinantiserum.

MATERIALS AND METHODSMaterials. Male Sprague-Dawley rats (125-150 g) were

obtained from Harlan Sprague Dawley (Indianapolis). Thepolypeptide hormones ovine, rat, and bovine prolactin andhuman, ovine, and rat growth hormone as well as antisera toovine and rat prolactin were obtained from the NationalHormone and Pituitary Program (National Institute of Dia-betes and Digestive and Kidney Diseases). Epidermal growthfactor (EGF), zinc-free insulin, 8-bromo-cAMP, sphingosine,PTA, and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine(H-7) were obtained from Sigma. Cyclosporine (Sandim-mune) was obtained from Sandoz. The monoclonal antibodyto the rat hepatic prolactin receptor (E29) was a generous giftfrom Paul A. Kelly (McGill University, Montreal).

Preparation of Liver Nuclei. Hepatic nuclei were preparedby using a modification of the technique of Widnell and Tata(25). Rats were killed by cervical dislocation. The liver wasperfused, washed, and minced in 2 vol ofice-cold bufferA (25mM Tris HCl, pH 7.4 at 40C/3 mM MgC12/0.32 M sucrose/2mM EGTA/0.1 mM spermine/0.1% Triton X-100 and leu-peptin at 50 ,ug/ml). The tissue was homogenized with a glassDounce homogenizer, filtered through two layers of nylonmesh, and diluted to 0.2 M sucrose. Buffer A was layeredbeneath the homogenate and the sample was centrifuged at

Abbreviations: PKC, protein kinase C; PTA, phorbol 12-tetra-decanoate 13-acetate; H-7, 1-(5-isoquinolinylsulfonyl)-2-methylpi-perazine; EGF, epidermal growth factor.

8649

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8650 Medical Sciences: Buckley et al.

700 x g for 10 min. The pellet was resuspended in a buffercontaining 2.4 M sucrose, 25 mM Tris HCl (pH 7.4 at 40C), 1mM MgCl2, 2 mM EGTA, 0.1 mM spermine, and 0.1% TritonX-100 and centrifuged at 50,000 x g for 60 min at 4TC. Thenuclear pellet was washed in a modified Hanks' balanced saltsolution (HBSS) containing 5 mM MgCl2 and resuspended at 1-5 x 107 nuclei per ml. Nuclear preparations were routinelyassessed for membrane contamination. These preparations hadno detectable 5'-nucleotidase activity (26) and had no contam-ination visible with light or electron microscopy (Fig. 1).For examination by electron microscopy, nuclear samples

were incubated overnight in 2.5% glutaraldehyde in a 0.1 Msodium phosphate buffer (pH 7.4), fixed in 2% osmiumtetroxide, dehydrated with acetone, and embedded in LX-112resin (Ladd, Burlington, VT). Sectioned samples (700 nm)were stained with uranyl acetate and lead citrate prior toexamination with a Phillips CM-10 transmission electronmicroscope (H. Lee Moffitt Cancer Center and ResearchInstitute, Department of Pathology, University of SouthFlorida).

Preparation of Splenocyte Nudei. Rats were killed bycervical dislocation. The spleens were forced through a finewire screen, rinsed in modified HBSS/0.3% bovine serumalbumin, and rocked for 10 min at room temperature. The cellsuspension was centrifuged at 300 x g for 5 min and the pelletwas resuspended in a pH 8.0 buffer containing 0.2 M NH4Cl,10 mM potassium carbonate, and 0.1 mM EDTA, invertedonce, filtered, and centrifuged at 300 X g for 5 min. Nucleiwere isolated from splenocytes resuspended in HBSS/0.3%bovine serum albumin by using the technique described byWolff et al. (27). The splenocyte suspension was centrifugedat 300 x g for 5 min, and resuspended at 1.5-3 x 107 cells perml in a buffer containing 0.4 mM potassium phosphate at pH6.7, 2 mM MgCl2, 0.1% Triton X-100, and 0.1 mM spermine.The sucrose concentration was adjusted to 0.32 M and thecells were centrifuged at 300 x g for 5 min. The pellet wasresuspended at 107 cells per ml in 0.32 M sucrose/4 mM

FIG. 1. Electron micrograph of a representative preparation ofisolated rat hepatic nuclei. (x6800.)

potassium phosphate, pH 6.7/2 mM MgCl2/4 mM EDTA/0.1mM spermine/0.1% Triton X-100/1 mM phenylmethanesul-fonyl fluoride/15 pug of leupeptin per ml and disrupted in aDounce homogenizer. The sample was centrifuged at 450 Xg for 10 min at 40C and the nuclear pellet was resuspended inmodified HBSS (108 nuclei per ml).

Incubation of Nuclei. Nuclei were incubated in 25-mlErlenmeyer flasks at 370C in a shaking water bath. Calciumwas added so that the final concentration was 1.0 mM. Thereaction was initiated by hormone addition and terminated bythe addition of 9 vol of ice-cold phosphate-buffered saline(Whittaker, Walkersville, MD) and centrifugation at 1300 xg for 20 min at 40C. The nuclei were resuspended in 1 ml ofbuffer B (25 mM Tris HCl, pH 7.4 at 40C/0.25 M sucrose/0.5mM EGTA/2 mM EDTA/2.5 mM MgCl2/50 mM 2-mercap-toethanol/0.5 mM phenylmethanesulfonyl fluoride/16 ,ug ofleupeptin per ml/0.02% Triton X-100) and disrupted bysonication (five 15-sec bursts with 5-sec rests). Purity of thenuclear preparation was verified by the absence of 5'-nucleotidase activity and by electron microscopy (Fig. 1).PKC Assay. Nuclear PKC was determined by using a

modification of the technique described by Kuo and cowork-ers (28) by measuring Ca2` and phospholipid-dependent phos-phorylation of lysine-rich histone (50 ,ug per assay, Sigma typeIII-s). The reaction in 20 mM Tris-HCl, pH 7.4/50 mM 2-mercaptoethanol/1 mM phenylmethylsulfonyl fluoride/8 mMMgCl2/0.5 mM CaCl2/12 ,ug of leupeptin per ml/10 ,M ATP(containing 106 cpm of [y-32P]ATP) with or without phospha-tidylserine at 25 ,ug/ml and dioleoylglycerol at 4 ,g/ml wasinitiated by the addition of 1-10 jig of protein in a total volumeof250 ,ul and was terminated by the addition of 3 ml of ice-cold25% trichloroacetic acid. Protein precipitate was collected byfiltration through Whatman GF/C filters. Filters were washedtwice with 3 ml of 10%6 trichloroacetic acid and dried and theirradioactivities were measured by liquid scintillation spectros-copy. Enzyme activity was determined by subtracting theamount of 32p incorporated into histone in the absence ofadded phospholipids from that incorporated in the presence ofphospholipids.

Statistical Determinations. Statistical differences betweentreatments were determined by the Newman-Keuls range formultiple comparisons, by analysis of variance (ANOVA), orby the Student t test for unpaired data. Differences wereconsidered significant at the 95% confidence level.

RESULTS AND DISCUSSIONTime Course for Prolactin Activation of PKC in Rat Liver

Nuclei. Within 1 min after the addition of prolactin, nuclearPKC activity was increased significantly (P < 0.01) comparedto vehicle controls (Fig. 2). Maximal prolactin activation ofnuclear PKC was found within 3 min (P < 0.01). The timecourse for prolactin activation ofPKC in nuclei isolated fromrat liver is nearly identical to that reported by Cambier et al.(29) for antibodies directed against Ia antigens and cyclicnucleotides on nuclear PKC in murine splenocytes. Theseauthors interpreted their results to reflect translocation ofPKC from the splenocyte cytosol to the nucleus. However,in the present study, hepatic nuclei were prepared free ofcontamination from either the cytosol or membrane (Fig. 1).Therefore, it appears that prolactin activated PKC present inthe hepatic nuclei.Dose Dependency of Prolactin-Stimulated Rat Liver Nuclear

PKC. Ovine prolactin at concentrations as low as 10-15 Mcaused detectable activation of PKC in rat liver nuclei (Fig.3). Ovine prolactin at 10-10 M consistently resulted in thegreatest activation of nuclear PKC. The activation by ratprolactin was somewhat less than ovine prolactin at 1010 M.However, rat prolactin was more potent than the ovinehomologue, stimulating nuclear PKC to greater than 130-fold

Proc. Natl. Acad. Sci. USA 85 (1988)

Proc. Natl. Acad. Sci. USA 85 (1988) 8651

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FIG. 2. Time course of prolactin stimulation of nuclear PKCactivity. Rat hepatic nuclei were incubated in the presence of 10-10M prolactin at 370C for the times indicated, after which PKC was

assayed in triplicate. A representative experiment is shown. Thisexperiment was repeated three times with equivalent results. All timepoints, with the exception of 20 min, differed significantly (P < 0.01)from controls (zero-time point).

vehicle controls at 10-12 M. Prolactin stimulation of nuclearPKC was biphasic, a characteristic previously described forprolactin effects in other systems (30-32).

Effect of Polypeptide Hormones and Other Biological Re-sponse Modifiers on PKC Activity in Isolated Rat Liver Nuclei.The ability of various prolactins and related growth hormonesto stimulate nuclear PKC was assessed (Table 1). Ovine, rat,and bovine prolactin significantly stimulated nuclear PKC.Human growth hormone, which recognizes both somatotro-pin and prolactin receptors in rat liver (33), also significantlystimulated PKC in the rat liver nucleus, whereas ovine andrat growth hormones were ineffective.

A

200

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PROLACTIN CONCENTRATION, log,. M

FIG. 3. Dose response for prolactin activation of PKC in rathepatic nuclei. Dose-dependent activation of nuclear PKC in re-

sponse to ovine (A) or rat (B) prolactin is presented. Rat hepaticnuclei were incubated at 370C for 3 min in the presence of hormoneat the concentration indicated on the abscissa. PKC was assayed intriplicate, and the data represent the mean ± SEM of three separateexperiments. All data points differed significantly (P < 0.01) from therespective vehicle control (CTL) with the exception of 10-7 M ratprolactin.

Table 1. Effect of various hormones and biological responsemodifiers on PKC activity in isolated rat liver nuclei

PKC, pmol phosphateTreatment per min per mg protein

Vehicle control 1 ± 0.8Ovine prolactin 229 ± 46*Rat prolactin (10-12 M) 134 ± 2*Bovine prolactin 91 ± 15*Human growth hormone 64 ± 3*Ovine growth hormone 1 ± 0.9EGF 37 ± 5*Ovine prolactin (10-11 M) 76 ± 11*EGF + ovine prolactin (10-1" M) 205 ± 21*Insulin 5 ± 2Dexamethasone 1 ± 0.7PTA 201 ± 35*8-Bromo-cAMP (10-' M) 1 ± 0.6

Rat liver nuclei were incubated for 3 min at 370C with the indicatedhormone or other substance at 10-10 M unless otherwise specified.PKC activity was measured in triplicate. Data represent the mean ±SEM of three separate experiments. *P < 0.01 vs. vehicle control.

EGF also significantly stimulated nuclear PKC (Table 1).This is of particular interest since EGF is mitogenic forhepatocytes maintained in culture (34, 35). However, whenEGF was added to hepatic nuclei in the presence of prolactinat a suboptimal concentration (10-11 M), there was a markedsynergistic response. These results suggest that while EGFcan stimulate nuclear PKC, it most likely increases PKC ofa different type or in a different subnuclear compartment.PTA activated nuclear PKC to a degree similar to that of

ovine prolactin (Table 1). This result is consistent with pre-vious observations showing that tumor-promoting phorbolesters mimic certain aspects of prolactin action in rat liver (3-5, 11) and in Nb2 node lymphoma cells (6, 15, 16, 36). Theability of tumor-promoting phorbol esters to activate PKCcorrelates positively with their efficacy as promoting agents(15, 37). Moreover, prolactin has been shown to promote thedevelopment of preneoplastic lesions in carcinogen-treatedrat liver (7). The demonstration now that both PTA andprolactin rapidly activate nuclear PKC suggests that nuclearactivation of this kinase represents a general characteristic oftumor-promoting substances.

Insulin, which is regarded as a hepatotrophic hormone (38,39), did not significantly activate nuclear PKC. Also, noactivation was detected in response to a cAMP analogue(Table 1). The synthetic corticosteroid dexamethasone hadno detectable effect on nuclear PKC activity.

Antibodies Directed Against Prolactin and the HepaticProlactin Receptor Inhibit Prolactin Activation of NuclearPKC. The effects of anti-ovine or anti-rat prolactin antiserumon prolactin-stimulated activation of nuclear PKC wereassessed in the presence of 10-10 M homologous prolactin(Table 2). In this experiment, each antiserum abolished theactivation of nuclear PKC by the respective prolactin. Thesedata demonstrate the specificity of prolactin for stimulationof nuclear PKC. Also shown in Table 2 is the effect of amonoclonal antibody to the rat hepatic prolactin receptor (40)on the activation of hepatic PKC. The antibody used in thisstudy (E29) inhibited binding of 125"-labeled prolactin by 95%when added at 200 ,ug/ml to either rat liver microsomes orsoluble prolactin receptors (40). Addition of the antibody at200 ,ug/ml abrogated the stimulatory effect of prolactin onnuclear PKC (Table 2). These data strongly suggest thatactivation of nuclear PKC by prolactin is a prolactin receptor-mediated phenomenon at the level of the nucleus. Further-more, the demonstration of the existence of several enzymesand substrates associated with diacylglycerol production inthe rat liver nucleus (41) and the recent report describing the

Medical Sciences: Buckley et al.

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8652 Medical Sciences: Buckley et al.

Table 2. Antibodies to prolactin and to the rat liver prolactinreceptor inhibit prolactin-dependent activation ofnuclear PKC

PKC, pmol phosphateTreatment per min per mg protein

Vehicle control 1 ± 0.8Ovine prolactin 185 ± 47*+ Antiserum to ovine prolactin 0.3 ± 0.2+ Antibody to rat prolactin receptor 0.2 ± 0.2

Rat prolactin 61 ± 7*+ Antiserum to rat prolactin 0.2 ± 0.1Rat liver nuclei were incubated for 3 min at 37TC in the presence

of ovine or rat prolactin (10-10 M) with or without the addition ofanti-prolactin antiserum (1:750 dilution) or the monoclonal antibodyto rat hepatic prolactin receptor (200 ,ug/ml). PKC activity wasdetermined in triplicate, and data represent the mean ± SEM ofthreeseparate experiments.*P < 0.01 vs. vehicle control.

synthesis of inositolphospholipids in nuclei of Friend cells(42) suggest that the biochemical machinery necessary foractivation of PKC is present in the nucleus.

Effect of Pharmacological Inhibitors on Prolactin-Stimu-lated ?KC Activation in Rat Liver Nucleus. Inhibitors ofPKCwere assessed for their effects on the activation of nuclearPKC by prolactin (Table 3). Low micromolar concentrationsof either H-7 or sphingosine inhibited the ability of prolactinto acti'vate PKC in the nucleus. Importantly, the addition ofcyclosponrne, an antagonist of prolactin binding and prolac-tin-stimulated macromolecular synthesis (4, 43, 44), com-pletely blocked activation of nuclear PKC by prolactin (Table3). The ability of PKC antagonists to block prolactin activa-tion of this nuclear kinase at concentrations shown to beeffective at inhibiting PKC in other systems (45, 46) demon-strates that PKC present in the rat liver nucleus is susceptibleto pharmacological manipulation in a similar manner to PKCpresent in other cellular compartments.

Nuclear Site oe Action for Prolactin in Tissues Where It HasMitogenic Action. The dramatic activation of hepatic nuclearPKC in response to prolactin or PTA suggests that other celltypes in which prolactin promotes mitogenic activity mightalso utilize a nuclear activation mechanism. Prolactin hasbeen implicated in the regulation of mitogenesis in lymphoidtissues. Prolactin administration to rats reverses hypophy-sectomy-induced immunosuppression (47-49). Human androdent lymphocytes have specific high-affinity prolactinreceptors that are blocked by cyclosporine (43, 44, 50-52).Recently it has been reported. by Russell and coworkers (50,53-56) that prolactin is required for mitogenesis in inurinesplenocytes. FUrther, murine splenocytes make and secretea 46-kDa lymphocyte prolactin in response to concanavalin Astimulation, and mitogenesis can be blocked totally by theaddition of anti-prolactin antiserum (55, 56). An explosion ofother reports corroborates the finding that prolactin modu-

Table 3. Pharmacological inhibitors of prolactin binding andPKC activation abolish lactogen-stimulated nuclear PKC activity

Inhibitor conc., PKC, pmol phosphateTreatment ;LM per min per mg protein

Vehicle control 1.0 ± 0.8Ovine prolactin 225 ± 14*+ H-7 6 28 ± 28+ Sphingosine 20 0.6 ± 0.5+ Cyclosporine 1 0.8 ± 0.6

Table 4. Prolactin activates nuclear PKC in tissues where it hasmitogenic action

Tissue PKC*

Rat liver nuclei 1.0 ± 0.6+ Ovine prolactin 230 ± 19t

Rat splenocyte nuclei 5 ± 2+ Ovine prolactin 717 ± 58t

Nuclei were incubated in the presence or absence of prolactin(10-10 M) for 3 min at 37TC. PKC activity was determined intriplicate, and the data are expressed as the mean ± SEM of threeseparate experiments.*PKC activity is expressed as pmol of phosphate per min per mg ofprotein for liver nuclei and as pmol of phosphate per min per 4 x10i nuclei for splenocyte nuclei.tp < 0.01 vs. respective vehicle control.

lates immunity in a variety ofimmune responses (44, 57-62).Therefore, it was important to test whether prolactin wouldactivate PKC in nuclear preparations of splenic mononuclearcells. The addition of 10-10 M prolactin to rat splenocytenuclei resulted in a dramatic elevation ofPKC activity within3 min (Table 4). Kuo and coworkers (28) report an exceed-ingly high level of PKC in spleen, even higher than in braintissue. The ability of prolactin to activate PKC in apparentlypure nuclear fractions of both liver and splenic mononuclearcells, tissues for which prolactin is a known dose-dependentmitogen, strongly suggests that PKC-mediated mitogenesis isregulated at the nuclear level by appropriate hormones andgrowth factors. Further, the synergistic ability of prolactinand EGF, both mitogenic agents for liver, to activate nuclearPKC provides an interaction mechanism for liver mitogenesissimilar to the platelet-derived growth factor/EGF/insulin-like growth factor I effects demonstrated for mitogenesis offibroblasts (63).The implications of this paper are important for our

understanding of nuclear function of polypeptide hormonessuch as prolactin. Previously, we reported that prolactin, aswell as partial hepatectomy, which has been linked to anincrease in circulating prolactin, caused an apparent trans-location ofPKC to the particulate fraction in rat liver (13). Inview of the present report, increased PKC activity in thishepatic fraction may reflect prolactin-mediated activation ofnuclear PKC, since nuclear components are present in the106,000 x g pellet in which PKC activity was assessed. Theability of PTA to mimic the effects of prolactin on nuclearPKC activation suggests that stimulation ofnuclear PKC maybe a general mechanism by which tumor-promoting sub-stances mediate a pleiotropic response. In our study, onlypolypeptide hormones that stimulate liver mitogenesis acti-vated nuclear PKC. This suggests that activation of nuclearPKC by internalized hormones or growth factors representsan initial signalling mechanism for phosphorylation of keynuclear proteins, resulting in new RNA and protein synthe-sis. Of further significance, the immunosuppressive cyclicpeptide cyclosporine completely inhibited prolactin activa-tion of nuclear PKC. We have hypothesized that the immu-nosuppressive properties of cyclosporine are related to itsability to antagonize prolactin action in lymphocytes, asystem in which prolactin serves as a comitogen (53-56). Thepresent report provides evidence that this agent may exert itsantiproliferative effects at the level of the cell nucleus, whereit interferes with prolactin receptor interactions. A nuclearsite ofaction for prolactin opens the door for numerous futurestudies of phospholipid metabolism, phosphorylation pat-terns, and new gene expression.

This study was supported by U.S. Public Health Service GrantsES-03587 and CA-48673 and by the University of South Florida(6113-930-RO).

Rat liver nuclei were incubated for 3 min at 37TC in the presenceof 10-10 M prolactin with or without the addition of the antagonistsindicated. PKC activity was determined in triplicate, and data areexpressed as the mean + SEM of three separate experiments.*P < 0.01 vs. vehicle control.

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Proc. Natl. Acad. Sci. USA 85 (1988) 8653

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